Biological control of diseases of harvested agricultural commodities using strains of the yeast candida oleophila

ABSTRACT

The present invention is drawn to isolates of Candida oleophila which are effective for the control of postharvest diseases in fruit and to biocontrol compositions which include such isolates. A method of utilizing the isolates to inhibit pathogens which cause postharvest diseases is also described. The organisms were isolated from the surface of tomato fruit and are useful for the control of a variety of fruit-rot pathogens in a variety of fruits.

This is a division of application Ser. No. 08/136,182, filed Oct. 15, 1993, now U.S. Pat. No. 5,425,941, which is a continuation-in-part of application Ser. No. 07/745,796, filed Aug. 16, 1991, now abandoned, herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the biological control of postharvest diseases in fruit. More particularly, this invention relates to a method for biologically controlling postharvest rots on fruit using strains of Candida oleophila (C. oleophila).

2. Description of the Prior Art

Postharvest diseases of fruit cause 15 to 25% losses yearly in the fruit industry worldwide. Fungicides, the major means for combating these diseases, are often ineffective and pose hazards to humans and the environment. Therefore, a critical need exists for new methods to control postharvest diseases.

Recently, it has been shown that the postharvest treatment of fruit with antagonistic microorganisms is an effective approach to the control of postharvest rot. Remarkable success was shown in the control of brown rot in peaches caused by Monilinia fructicola with the bacterium Bacillus subtilis (Pusey et al., Plant Dis., 86: 753-756, 1986), and the yeast Pichia guilliermondii has been shown to control a variety of postharvest rots of fruits and vegetables (Droby et al., Postharvest News and Information, 2: 169-173, 1991). A review of this subject can be found in Annual Reviews (Wilson and Wisniewski, Annual Review of Plant Pathology, 27: 425-441, 1989).

SUMMARY OF THE INVENTION

We have discovered that strains of C. oleophila are highly effective in controlling a variety of fruit-rot pathogens which affect several species of fruit. As a preferred embodiment, three isolates of the new strains have been deposited on Jun. 18, 1991 with the culture collection of the Northern Regional Research Center, U.S. Department of Agriculture, Peoria, Ill. 61604, under the acquisition numbers NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846. The deposited materials have been accepted for deposit under the Budapest Treaty on the international Recognition of the Deposit of Microorganisms for the purpose of patent procedures. Further, (1) said depository affords permanence of the deposits and ready accessibility thereto by the public if a patent is granted, (2) the materials have been deposited under conditions that assure that access to the material will be available during the pendency of the patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. All restrictions on the availability of progenies of the strain to the public will be irrevocably removed upon the granting of the patent. These isolates have been identified as Candida oleophila Montrocher by the Centraalbureau voor Schimmelcultures in the Netherlands and the German Collection of Microorganisms and Cell Cultures, Federal Republic of Germany.

Accordingly, it is an object of the present invention to provide a novel biological control agent which is safe and highly effective for the control of a variety of postharvest diseases in a variety of fruits.

It is also an object of the invention to provide a method of biologically controlling postharvest diseases in fruit which eliminates the use of fungicidal treatments.

In accordance with our invention, fruits are subjected to a composition comprised of at least one isolate of C. oleophila having the identifying characteristics of C. oleophila NRRL Y-NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846. In effect, the organisms multiply and occupy the surfaces of fruit wounds, thereby preventing infection by fruit-rot pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph of percent rot of apples treated with NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846 and water and challenged with the rot organisms Botrytis cinerea and Penicillium expansum.

FIG. 2 is a bar graph of percent rot of oranges treated with NRRL Y-18844, NRRL Y-18845 AND NRRL Y-18846 and oranges treated only with water, showing inhibition of Penicillium digitatum rot.

FIG. 3 is a bar graph showing total rot of peaches treated with Candida oleophila (NRRL Y-18846) on a semi-commercial packing line as compared with peaches receiving the standard fungicidal treatment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Isolates NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846 were obtained from the surface of tomato fruit by repeatedly washing the fruit with sterile water. The organisms are thereafter

                  TABLE 1                                                          ______________________________________                                                    Isolate Number                                                                 NRRL     NRRL     NRRL                                                         Y-18844  Y-18845  Y-18846                                           ______________________________________                                         GROWTH                                                                         D-glucose    +          +        +                                             D-galactose  -          -        +                                             L-sorbose    +          +        +                                             D-glucosamine                                                                               +          +        +                                             D-ribose     -          -        -                                             D-xylose     +.sup.W    +.sup.W  +                                             L-arabinose  -          -        -                                             L-rhamnose   -          -        -                                             sucrose      +          +        +                                             maltose      +          +        +                                             α,α trehalose                                                                   +          +        +                                             methyl α-glucoside                                                                    -          -        -                                             cellobiose   +          +        -                                             melibiose    -          -        -                                             lactose      -          -        -                                             raffinose    -          -        -                                             melezitose   +          +        +                                             glycerol     +          +        +                                             meso-erythritol                                                                             -          -        -                                             D-glucitol   +          +        +                                             myo-inositol -          -        -                                             2-keto-D-gluconate                                                                          +          +        +                                             D-gluconate  -          -        -                                             D-glucuronate                                                                               -          -        -                                             DL-lactate   -          -        -                                             FERMENTATION                                                                   glucose      +          +        +                                             galactose    -          -        +                                             maltose      -          -        -                                             sucrose      .sup.vw    .sup.vw  -                                             lactose      -          -        -                                             raffinose    -          -        -                                             nitrate      -          -        -                                             ethylamine   +          +        +                                             L-Lysine     +          +        +                                             cadaverine   +          +        +                                             TEMPERATURE  -          -        -                                             at 37° C.                                                               ANTIBIOTICS                                                                    .01% cycloheximide                                                                          +.sup.W    -        +                                             ______________________________________                                          .sup.w = weak;                                                                 .sup.vw = very weak                                                      

plated and grown on any nutritionally rich medium sufficient to support growth of microorganisms. Preferably, the medium is either yeast dextrose agar (NYDA) or yeast-malt extract agar (YM).

Isolates NRRL Y-18844, NRRL 18845 and NRRL Y-18846 have the following identifying characterisitcs: colonies are cream white, slightly raised, shiny, round and smooth. Pseudohyphae were observed. Characteristics observed during biochemical and phsiological tests are presented in Table 1.

Growth of the organisms is effected under aerobic conditions at any temperature satisfactory for growth of the organism; i.e. from about 10° C. to about 30° C. The preferred temperature range is about 20° C. to 25° C. The pH of the nutrient medium is about neutral; i.e. 6.7 to 7.2. The incubation time is that time necessary for the organisms to reach a stationary phase of growth. Incubation time is preferably from about 40 to 60 hours.

Isolates NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846 may be grown in any conventional shake flask for small fermentation runs. For large scale operations, it is convenient to carry out the culture in a fermentation tank while applying agitation and aeration to the inoculated liquid medium. Following incubation, the organisms are harvested by conventional sedimentary methodology; i.e. centrifugation or filtration. Cultures are stored on silica gel and frozen until used.

Isolates NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846 are useful for the control of a variety of fruit-rot pathogens, including but not limited to, Penicillium digitatum, Botrytis cinerea, Rhizopus stolinifer, Penicillium expansum and Monilinia fructicola.

The organisms of the invention are useful for the control of postharvest diseases in a variety of fruits including, but not limited to, all cultivars of-citrus fruits, grapes, apples, pears, tomatoes, persimmons and the like. Suitable citrus fruits include, but are not limited to, grapefruits, oranges, lemons and the like.

The organisms of the invention are preferably applied to the fruits in suspension with water or any other agriculturally acceptable excipient such as dusts. Compositions comprising natural plant oils such as corn oil and cottonseed oil are effective, and preparations resulting in powders for dusting are particularly useful since the steps required for wetting and drying the fruit are eliminated. When the organisms are grown in a liquid medium, they may be applied in suspension with the liquid medium. Suspensions of the organisms of the invention may also include conventional additives such as surfactants and wetting agents to enhance the effectiveness of the organisms. As an integrated approach, the organisms of the present invention may be used with a very low concentration of a fungicide.

Useful concentrations of suspensions are any concentrations which inhibit the development of the targeted fruit-rot pathogen when applied to the fruit. As will be obvious to one skilled in the art, effective concentrations may vary depending upon such factors as: (1) the type of fruit; (2) the ripeness of the fruit; (3) the concentration of pathogens affecting the fruit; (4) the type of wound on the fruit; (5) temperature and humidity; and (6) the age of the fruitsrot pathogen. Exemplary concentrations range from about 1×10⁴ to 1×10⁹ CFU/ml, most preferably, from about 1×10⁷ to 1×10⁹ CFU/ml. For purposes of the invention, the abbreviation "CFU" is used herein to designate "colony forming units".

The organisms of the invention may be applied to fruit using conventional methods such as dipping, spraying, or brushing. In addition, the organisms of the invention may be incorporated into waxes, wraps or other protective coatings used in processing the fruit.

The fruit may be treated anytime before or after harvest. Typically, the preferred time of treatment is after harvest and prior to storage or shipment.

It is within the scope of the invention to treat the fruit with isolates NRRL Y-18844, NRRL Y-18845 or NRRL Y-18846, alone or in combination (i.e., C. oleophila having the identifying characteristics of C. oteophila NRRL Y-18844, NRRL Y-18845 or NRRL Y-18846 alone or a mixture of two or more such isolates). The organisms may also be used in combination with other control agents useful for inhibiting the development of fruit-rot pathogens on fruit. When used, these agents should be used in an amount, as readily determined by one skilled in the art, which will not interfere with the effectiveness of the organisms of the invention.

The following examples are intended to further illustrate the invention and not to limit the scope of the invention as defined by the claims.

EXAMPLE I

The effectiveness of C. oleophila (NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846) was evaluated using "Red Delicious" apples. The fruit rot pathogens tested were Botrytis cinerea and Penicillium expansum, responsible for gray mold and green mold.

Biologically pure cultures of Candida oleophila isolates were obtained by using the following procedures: a tomato was dipped in a container of 150 ml sterile deionized water, and the fruit was washed two separate times. Five apples were wounded (3 mm×5 mm deep). Fifty μl of the first and second washing solution was placed in each wound. After 2 hours the wounds were inoculated with 20 μl of a spore suspension of Botrytis cinerea (1×10⁵ CFU/ml). The fruit were stored in trays at room temperature.

After 10 days, wounds on the inoculated apples not showing rot were scraped with a sterile inoculating needle. Dislodged material was diluted in 10 ml of sterile water, and 100 μl of this suspension was spread on yeast-maltose agar plates. After 1 day, single colonies with different visual appearance were streaked on plates of yeast-maltose agar. After the colonies developed, each was checked microscopically to separate the yeasts and bacteria.

Three isolates of Candida oleophila were derived by the method (NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846). These isolates were grown in a liquid yeast maltose medium for 24 hours, and 50 μl of a 1×10⁸ CFU suspension of washed cells was pipetted into apple wounds. After 2 hours, the treated wounds were inoculated with 20 μl of Botrytis cinerea or Penicillium expansum spores (1×10⁵ spores/ml). After 14 days the wounds were examined for rot. FIG. 1 summarizes the results. All three isolates effectively controlled both Botrytis and Penicillium rots of apple.

EXAMPLE II

The effectiveness of three isolates of C. oleophila (NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846) for inhibiting Penicillium digitatum on oranges was tested.

Oranges (Citrus sinesis) were washed with 2% sodium hypochlorite to surface sterilize the fruit. After air drying, the oranges were placed on styrofoam trays in plastic trays with lids. Water (100 ml) was added to each tray for humidity. The oranges were wounded using a needle. Wound size was 4 mm wide by 5 mm deep. Three-day-old shake cultures of NRRL Y-18844, NRRL Y-18845 and NRRL Y-18846 growing on NYDB at a 1×10⁹ CFU/ml concentration were added to the wound, 50 μl/wound, and the oranges were allowed to air dry. Thereafter an aqueous suspension of Penicillium digitatum (1×10⁴ spores/ml) were added to the wounds, 20 μl/wound. Controls were inoculated with water only.

All three isolates of C. oleophila reduced rot caused by Penicillium digitatum as shown in FIG. 2.

EXAMPLE III

The effectiveness of C. oleophila isolate NRRL Y-18846 was compared with commercial fungicides for the control of Monilinia fructicola and Rhizopus stolinfer rots of Candor peaches as postharvest treatments on a semi-commercial packing line. Ten boxes per treatment of peaches (64 peaches per box) were placed on a semi-commercial peach processing line where they were (1) washed and defuzzed; (2) dried; (3) treated with a micronized spray of the antagonist C. oleophila (isolate NRRL Y-18846); (4) dried; and (5) waxed.

Four treatments were compared: (1) peaches waxed and not treated with the commercial rate of the fungicides TOPSIN (4,4'-O-phenylene bis 3-thioallophanate) and BOTRAN (2,6-dichloro-4-nitroaniline) (2) peaches waxed with a preparation which contained the commercial rate of the fungicides TOPSIN (diethyl 4,4'-O-phenylene bis 3-thioallophanate) and BOTRAN (2,6-dichloro-4-nitroaniline) (1 lb/100 gallons); (3) peaches washed but not waxed or treated with fungicides; and (4) peaches washed, dried, treated with a micronized preparation of C. oleophila isolate (NRRL Y-18846), dried and waxed.

Following the treatments the peaches were placed in trays and stacked in boxes where they were stored at 10° C. Every 4 days for 16 days the number of rotted fruit was recorded. The results are presented in FIG. 3.

NRRL Y-18846 effectively controlled both Monilia and Rhizopus rots of peaches when applied as a micronized spray on a peach processing line. It provided better protection against rot than commerical fungicide treatments or wax alone.

The foregoing detailed descriptions and examples are given merely for purposes of illustration. Modifications and variations may be made therein without departing from the spirit and scope of the invention. 

We claim:
 1. A biologically pure culture of Candida oleophila effective for the inhibition of fruit-rot pathogens, where Candida oleophila is NRRL Y-18844, NRRL Y-18845 or NRRL Y-18846.
 2. The biologically pure culture of claim 1, wherein Candida oleophila is NRRL Y-18844.
 3. The biologically pure culture of claim 1, wherein Candida oleophila is NRRL Y-18845.
 4. The biologically pure culture of claim 1, wherein Candida oleophila is NRRL Y-18846.
 5. A biocontrol composition comprising Candida oleophila effective for the inhibition of fruit-rot pathogens and an agriculturally acceptable excipient, wherein Candida oleoplila is NRRL Y-18844, NRRL Y-18845 or NRRL Y-18846.
 6. The biocontrol composition of claim 5, wherein Candida oleophila is NRRL Y-18844.
 7. The biocontrol composition of claim 5, wherein Candida oleophila is NRRL Y-18845.
 8. The biocontrol composition of claim 5, wherein Candida oleophila is NRRL Y-18846.
 9. The biocontrol composition of claim 5, wherein Candida oleophila is a combination of at least two of NRRL Y-18844, NRRL Y-18845 or NRRL Y-18846.
 10. The biocontrol composition of claim 6, wherein the agriculturally acceptable excipient is water, natural plant oils or dust.
 11. The biocontrol composition of claim 5, wherein the fruit-rot pathogen is Penicillium digitatum, Botrytis cinerea, Rhizopus stolinifer, Penicillium expansum, or Monilinia fructicola. 